Generation of Slco1a4-CreERT2-tdTomato Knock-in Mice for Specific Cerebrovascular Endothelial Cell Targeting.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood-brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

Glycosylation as a tracer of off-target Cre-lox activation in development.

The Cre-lox system is one of the most widely used methods for lineage-specific and inducible genome editing in vivo. However, incomplete penetrance and off-target effects due to transient promoter expression in a stem or pluripotent precursor cell can be problematic and difficult to detect, especially if the target gene is not normally present in the fully differentiated but off-target cells. Yet, the loss of the target gene through the transient expression of Cre may impact the differentiation of those cells by virtue of transient expression in a precursor population. In these situations, off-target effects in an unknown precursor cell can, at best, complicate conclusions drawn from the model, and at worst, invalidate all data generated from that knockout strain. Thus, identifying Cre-driver promoter expression along entire cell lineages is crucial to improve rigor and reproducibility. As an example, transient expression in an early precursor cell has been documented in a variety of Cre strains such as the Tie2-based Cre-driver system that is used as an "endothelial cell-specific" model 1. Yet, Tie2 is now known to be transiently expressed in a stem cell upstream of both hematopoietic and endothelial cell lineages. Here, we use the Tie2 Cre-driver strain to demonstrate that due to its ubiquitous nature, plasma membrane glycans are a useful marker of both penetrance and specificity of a Cre-based knockout.

Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow.

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.

Comparison of Integrase Strand Transfer Inhibitors (INSTIs) and Protease-Boosted Inhibitors (PIs) on the Reduction in Chronic Immune Activation in a Virally Suppressed, Mainly Male Population Living with HIV (PLWH).

Background and Objectives: The success of combined antiretroviral therapy (cART) has led to a dramatic improvement in the life expectancy of people living with HIV (PLWH). However, there has been an observed increase in cardiometabolic, bone, renal, hepatic, and neurocognitive manifestations, as well as neoplasms, known as serious non-AIDS events/SNAEs, compared to the general population of corresponding age. This increase is linked to a harmful phenomenon called inflammaging/immunosenescence, which is driven by chronic immune activation and intestinal bacterial translocation. In this study, we examined immunological and metabolic parameters in individuals receiving current cART. Materials and Methods: The study was conducted at Laiko General Hospital in Athens, Greece. Plasma concentrations of sCD14, IL-6, SuPAR, I-FABP, and LBP were measured in virally suppressed PLWH under cART with at least 350 CD4 lymphocytes/µL. We compared these levels between PLWH receiving integrase strand transfer inhibitors (INSTIs) and protease inhibitors (PIs) and attempted to correlate them with chronic immune activation and metabolic parameters. Results: Data from 28 PLWH were analyzed, with a mean age of 52 and 93% being males. Among the two comparison groups, IL-6 levels were higher in the PIs group (5.65 vs. 7.11 pg/mL, p = 0.03). No statistically significant differences were found in the other measured parameters. A greater proportion of PLWH under INSTIs had normal-range LBP (33% vs. 0%, p = 0.04). When using inverse probability of treatment weighting, no statistically significant differences in the measured parameters were found between the two groups (sCD14 p = 0.511, IL-6 p = 0.383, SuPAR p = 0.793, I-FABP p = 0.868, and LBP p = 0.663). Glucose levels were found to increase after viral suppression in the entire sample (92 mg/dL vs. 98 mg/dL, p = 0.009). Total (191 mg/dL vs. 222 mg/dL, p = 0.005) and LDL cholesterol (104 mg/dL vs. 140 mg/dL, p = 0.002) levels were higher in the PIs group. No significant differences were observed in liver and renal function tests. Conclusions: Further investigation is warranted for PLWH on cART-containing INSTI regimens to explore potential reductions in chronic immune activation and intestinal bacterial translocation.

Variegate expression of Cre recombinase in hematopoietic cells in CD11c-cre transgenic mice.

In this study, we performed an in-depth analysis of Cre expression in the widely used CD11c-Cre transgenic mice generated by the group of Boris Reizis. In contrast to previous observation, using the highly sensitive Rosa-26-floxed-tdTomato reporter mouse line, we show variegated expression of Cre in multiple hematopoietic linage cells starting in hematopoietic stem cells. Indeed, we found that in the CD11c-Cre driver mice, Cre is expressed in cDC linage cells and pDC starting from the myeloid dendritic cell precursor, as expected, but also in a substantial fraction of hematopoietic stem cells and common lymphoid progenitors and, consequently, in >50% of all leukocytes. Hence, this study indicates that the reporter mice used to characterize Cre expression in Cre-driver mice should be selected with caution and considering the sensitivity of the reporter system. This study also suggests that the interpretation of some reports using this CD11c-Cre transgenic mice may need to be re-considered based on a careful evaluation of the cell type-specificity of Cre-mediated in their model.

Towards a Light-mediated Gene Therapy for the Eye using Caged Ethinylestradiol and the Inducible Cre/lox System.

Increasingly, retinal pathologies are being treated with virus-mediated gene therapies. To be able to target viral transgene expression specifically to the pathological regions of the retina with light, we established an in vivo photoactivated gene expression paradigm for retinal tissue. Based on the inducible Cre/lox system, we discovered that ethinylestradiol is a suitable alternative to Tamoxifen as ethinylestradiol is more amenable to modification with photosensitive protecting compounds, i.e., "caging." Identification of ethinylestradiol as a ligand for the mutated human estradiol receptor was supported by in silico binding studies showing the reduced binding of caged ethinylestradiol. Caged ethinylestradiol was injected into the eyes of double transgenic GFAP-CreERT2 mice with a Cre-dependent tdTomato reporter transgene followed by irradiation with light of 450 nm. Photoactivation significantly increased retinal tdTomato expression compared to controls. We thus demonstrated a first step towards the development of a targeted, light-mediated gene therapy for the eyes.

Polypharmacy and potential drug-drug interactions among people living with HIV in the era of integrase strand transfer inhibitor-based antiretroviral therapy.

OBJECTIVES: To investigate the prevalence of polypharmacy and potential drug-drug interactions (DDIs), and the factors associated with DDIs among people living with human immunodeficiency virus (HIV; PLWH) in the modern era of antiretroviral therapy (ART). METHODS: This cross-sectional study included PLWH who had been on ART for ≥3 months at two designated HIV hospitals in Taiwan. All ART and non-ART prescriptions were collected from the NHI-MediCloud System and screened for DDIs using the University of Liverpool HIV drug interactions database. A case-control analysis was conducted to investigate the factors associated with DDIs. RESULTS: In total, 1007 PLWH were included in this study from June 2021 to August 2022. The median age was 40 (interquartile range 33-49) years, and 96.2% were taking integrase strand transfer inhibitor (INSTI)-based ART. The proportions of PLWH with at least one non-communicable disease and polypharmacy were 50.0% and 18.7%, respectively. Seven (0.7%) PLWH had red-flagged DDIs, and 159 (15.8%) had amber-flagged DDIs. In multi-variable models, the prevalence of DDIs was associated with older age [adjusted odds ratio (aOR) per 1-year increase 1.022), number of co-medications (aOR 1.097), use of boosted INSTI-based ART (vs unboosted INSTI, aOR 8.653), and concomitant medications in the alimentary tract and metabolism category (aOR 11.058) and anti-neoplastic and immunomodulating agents (aOR 14.733). CONCLUSIONS: In the INSTI era, the prevalence of potential DDIs is lower than noted previously, but remains substantial. Clinicians should monitor DDIs routinely, especially in older PLWH, those taking a higher number of co-medications, and those who are taking booster-containing ART or medications from specific categories.

Integrase strand-transfer inhibitor use and cardiovascular events in adults with HIV: an emulation of target trials in the HIV-CAUSAL Collaboration and the Antiretroviral Therapy Cohort Collaboration.

BACKGROUND: A recent observational study suggested that the risk of cardiovascular events could be higher among antiretroviral therapy (ART)-naive individuals with HIV who receive integrase strand-transfer inhibitor (INSTI)-based ART than among those who receive other ART regimens. We aimed to emulate target trials separately in ART-naive and ART-experienced individuals with HIV to examine the effect of using INSTI-based regimens versus other ART regimens on the 4-year risk of cardiovascular events. METHODS: We used routinely recorded clinical data from 12 cohorts that collected information on cardiovascular events, BMI, and blood pressure from two international consortia of cohorts of people with HIV from Europe and North America. For the target trial in individuals who had previously never used ART (ie, ART-naive), eligibility criteria were aged 18 years or older, a detectable HIV-RNA measurement while ART-naive (>50 copies per mL), and no history of a cardiovascular event or cancer. Eligibility criteria for the target trial in those with previous use of non-INSTI-based ART (ie, ART-experienced) were the same except that individuals had to have been on at least one non-INSTI-based ART regimen and be virally suppressed (≤50 copies per mL). We assessed eligibility for both trials for each person-month between January, 2013, and January, 2023, and assigned individuals to the treatment strategy that was compatible with their data. We estimated the standardised 4-year risks of cardiovascular events (myocardial infarction, stroke, or invasive cardiovascular procedure) via pooled logistic regression models adjusting for time and baseline covariates. In per-protocol analyses, we censored individuals if they deviated from their assigned treatment strategy for more than 2 months and weighted uncensored individuals by the inverse of their time-varying probability of remaining uncensored. The denominator of the weight was estimated via a pooled logistic model that included baseline and time-varying covariates. FINDINGS: The analysis in ART-naive individuals included 10 767 INSTI initiators and 8292 non-initiators of INSTI. There were 43 cardiovascular events in INSTI initiators (median follow-up of 29 months; IQR 15-45) and 52 in non-initiators (39 months; 18-47): standardised 4-year risks were 0·76% (95% CI 0·51 to 1·04) in INSTI initiators and 0·75% (0·54 to 0·98) in non-INSTI initiators; risk ratio 1·01 (0·57 to 1·57); risk difference 0·0089% (-0·43 to 0·36). The analysis in ART-experienced individuals included 7875 INSTI initiators and 373 965 non-initiators. There were 56 events in INSTI initiators (median follow-up 18 months; IQR 9-29) and 3103 events (808 unique) in non-INSTI initiators (26 months; 15-37) in non-initiators: standardised 4-year risks 1·41% (95% CI 0·88 to 2·03) in INSTI initiators and 1·48% (1·28 to 1·71) in non-initiators; risk ratio 0·95 (0·60 to 1·36); risk difference -0·068% (-0·60 to 0·52). INTERPRETATION: We estimated that INSTI use did not result in a clinically meaningful increase of cardiovascular events in ART-naive and ART-experienced individuals with HIV. FUNDING: National Institute of Allergy and Infectious Diseases and National Institute on Alcohol Abuse and Alcoholism.

DNA strand breaks and gaps target retroviral intasome binding and integration.

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.

Cre-loxP System-Based Mouse Model for Investigating Graves' Disease and Associated Orbitopathy.

Background: Graves' disease (GD), one of the most common forms of autoimmune thyroid disorders, is characterized by hyperthyroidism caused by antibodies (Abs) against the extracellular A-subunit of the thyrotropin receptor (TSHR). Various approaches have been used to create mouse models of GD, including transfected fibroblasts and immunization with plasmids or adenoviruses expressing human TSHR A-subunit (hTSHR A-subunit). These models, however, require repeated immunization and produce inconsistent results. In this study, we established a novel Cre-loxP system-based mouse model that is able to generate the hTSHR A-subunit, mimicking human GD, and characterized the histological changes in Graves' orbitopathy (GO) progression after a single injection. Materials and Methods: A Cre-loxP system-based mouse model was constructed by inserting the CAG-loxP-STOP-loxP-hTSHR A-subunit cassette into the Rosa26 locus of the mouse genome. Conditional expression of the hTSHR A-subunit was successfully achieved by intramuscular injection of the transactivator of transcription-Cre recombinase (GD mice). Blood tests for anti-TSHR Abs and the total thyroxine (T4) level were performed. Magnetic resonance imaging (MRI) was used to monitor morphological changes in the eyes. A histological examination of the thyroid gland and retrobulbar tissues was performed to observe pathological changes. Results: Twenty-four (8 control and 16 GD) mice were investigated. All GD mice exhibited higher levels of TSHR Abs compared with the control group. Moreover, more than 80% of the mouse models showed elevated T4 levels accompanied by thyroid goiter. MRI analysis revealed an increased volume of retrobulbar tissue, while immunohistochemical staining of orbital tissues exhibited macrophage infiltration and muscle fibrosis in the GD mice, contrasting with the control group. Conclusions: Our novel mouse model for GD, which showed the histological features of GO, was successfully established using the Cre-loxP system. This animal model offers improved insights and contributes to advancing methodological developments for GD and GO.

G9a inactivation in progenitor cells with Isl1-Cre with reduced recombinase activity models aspects of Dandy-Walker complex.

G9a, also known as EHMT2, is essential for embryogenesis and has specific functions in multiple developmental processes. G9a inactivation affects development of the nervous system, which is formed with contribution of descendants of progenitor cells expressing the transcription factor Isl1. However, the function of G9a in Isl1-expressing progenitors is unknown. Here, we show that G9a is required for proper development of multiple structures formed with contribution of Isl1-expressing progenitors. A Cre-dependent GFP reporter revealed that the recombinase activity of the Isl1-Cre used in this study to inactivate G9a was reduced to a subset of Isl1-expressing progenitor cells. G9a mutants reached endpoint by 7 weeks of age with cardiac hypertrophy, hydrocephalus, underdeveloped cerebellum and hind limb paralysis, modeling aspects of Dandy-Walker complex. Moreover, neuroepithelium of the lateral ventricle derived from Isl1-expressing progenitors was thinner and disorganized, potentially compromising cerebrospinal fluid dynamics in G9a mutants. Micro-computed tomography after iodine staining revealed increased volume of the heart, eye lens and brain structures in G9a mutant fetuses. Thus, altered development of descendants of the second heart field and the neural crest could contribute to multicomponent malformation like Dandy-Walker.

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Neuroanatomical characterization of the Nmu-Cre knock-in mice reveals an interconnected network of unique neuropeptidergic cells.

Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. Although the central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under the Nmu promoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions, in situ hybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete characterization of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide.

INTRODUCTION: The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. MATERIALS AND METHODS: We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. RESULTS: We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. CONCLUSION: 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Warning regarding hematological toxicity of tamoxifen activated CreERT2 in young Rosa26CreERT2 mice.

The Cre-lox system is a versatile and powerful tool used in mouse genetics. It allows spatial and/or temporal control of the deletion of a target gene. The Rosa26-CreERT2 (R26CreERT2) mouse model allows ubiquitous expression of CreERT2. Once activated by tamoxifen, CreERT2 will enter into the nuclei and delete floxed DNA sequences. Here, we show that intraperitoneal injection of tamoxifen in young R26CreERT2 mice leads to morbidity and mortality within 10 days after the first injection, in the absence of a floxed allele. Activation of CreERT2 by tamoxifen led to severe hematological defects, with anemia and a strong disorganization of the bone marrow vascular bed. Cell proliferation was significantly reduced in the bone marrow and the spleen resulting in the depletion of several hematopoietic cells. However, not all cell types or organs were affected to the same extent. We realized that many research groups are not aware of the potential toxicity of Cre recombinases, resulting in misinterpretation of the observed phenotype and in a waste of time and resources. We discuss the necessity to include tamoxifen injected CreERT2 controls lacking a floxed allele in experimental designs and to improve communication about the limitations of Cre-lox mouse models among the scientific community.

Molecular determinants for Rous sarcoma virus intasome assemblies involved in retroviral integration.

Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their assembly process. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate at 3.36 Å resolution. The conserved intasome core region consisting of IN subunits contributing active sites interacting with viral/target DNA has a resolution of 3 Å. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the conserved intasome core, similar to results previously shown with the RSV octameric cleaved synaptic complex intasome produced with IN and viral DNA only. An extensive analysis of higher resolution STC structure helped in the identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246, and S124 in cleaved synaptic complex and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

Inducible Expression of Amphinase in Neuroblastoma Cells using Cre/LoxP System.

PURPOSE: To induce the expression of Amphinase, an antitumor ribonuclease from Rana pipiens oocytes, in neuroblastoma cell lines and build a foundation for mechanism study. METHODS: A loxP-cassette vector was constructed comprising a sequence of loxP -Puro-3∗polyA-loxP, followed by amphinase cDNA. The vector was transfected into neuroblastoma cell lines, SK-N-BE(2)-C, by Lipofectamine LTX. The transfected cells were selected by puromycin for two weeks. Polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) were conducted to verify that the loxP-cassette vector was stably transfected. The expression of amphinase was activated by the addition of Cre recombinase delivered by a lentiviral vector and identified by qPCR and Western blotting (WB). CCK8 assay and colony formation assay were conducted to check the effect of amphinase on cell proliferation. RNA sequencing (RNA-seq) was conducted to explore the targeted pathway of Cre/loxP-mediated amphinase and recombinant amphinase. RESULTS: Stably transfected cell clones were achieved through puromycin selection. After Cre recombinase was delivered to the cells, the loxP-flanked fragment was deleted and the expression of amphinase was induced, which were tested by PCR and qPCR. It was shown that cell proliferation was significantly inhibited by the amphinase mediated by the Cre/loxP system. KEGG enrichment and GSEA analysis indicated that amphinase had an impact on the ER function of neuroblastoma cells, which was identical to the effect of the recombinant amphinase. CONCLUSION: We successfully induce the expression of amphinase in neuroblastoma cell lines via Cre/loxP system. The Cre/loxP-mediated amphinase had a similar antitumor mechanism to the recombinant amphinase, providing a powerful tool for the mechanism study of amphinase.

A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages.

Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.

Flt3- and Tie2-Cre tracing identifies regeneration in sepsis from multipotent progenitors but not hematopoietic stem cells.

In response to infections and stress, hematopoiesis rapidly enhances blood and immune cell production. The stage within the hematopoietic hierarchy that accounts for this regeneration is unclear under natural conditions in vivo. We analyzed by differentiation tracing, using inducible Tie2- or Flt3-driven Cre recombinase, the roles of mouse hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). During polymicrobial sepsis, HSCs responded transcriptionally and increased their proliferation and cell death, yet HSC differentiation rates remained at steady-state levels. HSC differentiation was also independent from the ablation of various cellular compartments-bleeding, the antibody-mediated ablation of granulocytes or B lymphocytes, and genetic lymphocyte deficiency. By marked contrast, the fate mapping of MPPs in polymicrobial sepsis identified these cells as a major source for accelerated myeloid cell production. The regulation of blood and immune cell homeostasis by progenitors rather than stem cells may ensure a rapid response while preserving the integrity of the HSC population.